Advances are being made in technologies to efficiently analyze the genetic functions of a variety of living organisms. To analyze the expression of their genes or the nucleotide sequences of the genes, a detection tool called a DNA chip is used, in which a number of nucleic acid fragments are fixed on the surface of a solid support. The nucleic acid fragments bound or fixed on the surface of the solid support are also called probe nucleic acid fragments. A typical DNA chip is a microarray wherein a number of probe nucleic acid fragments are arrayed and fixed on a solid support such as a slide glass. The DNA chip related technologies relating to the production of the DNA chip and its use are believed to be applicable also to the detection of biomolecules other than DNA. Thus, there are expectations that such technologies will provide new means for the research of new drugs and the development of a method for diagnosing and preventing diseases.
A breakthrough in the progress of DNA-chip related technologies was achieved when a method of determining the nucleotide sequence of nucleic acid fragments based on hybridization with an oligonucleotid was developed. Even though this method can overcome the limitations in the methods of determining nucleotide sequences using gel electrophoresis, it was only later that the hybridization method came to be used for practical purposes.
Then, a DNA chip of the above-described structure and technologies for the production thereof were developed, and it became possible to examine the expression, mutation, polymorphism or the like of a gene efficiently in a short time. Specifically, a target nucleic acid fragment showing complementarity with the probe nucleic acid fragments on the produced DNA chip is generally detected by utilizing hybridization between the probe nucleic acid fragment on the DNA chip and the target nucleic acid fragment.
One method for detecting hybridization between the probe nucleic acid fragment on the DNA chip and the target nucleic acid fragment is a method of labeling the target nucleic acid fragment with a detectable molecule in advance. Most generally, the target nucleic acid is labeled with a fluorescent dye as the labeling molecule.
Following the hybridization process, fluorescence emitted from the surface of the DNA chip is measured to detect only those locations (spots) on the DNA chip where hybridization between the probe nucleic acid fragments and the target nucleic acid fragment occurred. It is also possible to determine the existing amount of target nucleic acid fragments on the basis of the intensity of the measured fluorescence. However, this method requires that the target nucleic acid be labeled by the fluorescent dye in advance. The measurement of the fluorescence emitted from the surface of the DNA chip has a disadvantage in that it is not a simple method, since a large-sized apparatus is required and the measurement takes time.
Another method of detecting hybridization between the probe nucleic acid fragment on the DNA chip and the target nucleic acid fragment is known from Japanese Patent No.2573443. This method detects hybridization by electrochemical measurement, using a double-stranded nucleic acid fragment recognizing substance which has an electrochemical activity and can bind specifically to the double-stranded nucleic acid. This method is simpler and superior in that it does not require the target nucleic acid fragment to be labeled in advance and in that the electrochemical measurement can be performed in a small-sized apparatus in a short time. The method is therefore expected to provide a new hybridization detecting means which can be used in the field of clinical examination, for example.
As described above, in the detection methods by utilizing hybridization between the probe nucleic acid fragment on the DNA chip and the target nucleic acid fragment, the reproducibility of the detection of the target nucleic acid fragment is dependent on whether or not the probe nucleic acid fragments are fixed on the surface of the solid support in a stable manner. Further, the fixing density of the probe nucleic acid fragments on the solid support surface (i.e., the amount of the same kind of probe nucleic acid fragments fixed per unit area) determines the sensitivity and limit with which the target nucleic acid can be detected. Thus, in order to realize a practical method of detecting a target nucleic acid by using a DNA chip, a technology must be provided for fix a number of probe nucleic acid fragments on the surface of a solid support in a stable and density-controlled manner.
A method of producing a DNA chip is known whereby an oligonucleotide is synthesized directly on the solid support surface (“on-chip method”). Another method involves the bonding and fixing of probe nucleic acid fragments prepared in advance on the surface of a solid support. A typical on-chip method is based on the use of a combination of a protection group which is selectively removed by irradiation of light, and photolithography and solid-phase synthesizing techniques which are used in the production of semiconductor, whereby an oligonucleotide is selectively synthesized in a predetermined small matrix region.
As methods of binding or fixing a probe nucleic acid fragment prepared in advance on the surface of a solid support, the following are known depending on the type of the probe nucleic acid fragment and the solid support.
(1) In the case where the fixed probe nucleic acid fragment is cDNA (complementary DNA synthesized by using mRNA as a template) or a PCR product (a DNA fragment obtained by amplifying cDNA by PCR), the cDNA or PCR product is spotted onto the surface of a solid support treated with a poly-cationic compound (e.g. polylysine or polyethylene-imine) so that the cDNA or PCR product is bound to the support via electrostatic bonding by utilizing the electric charge of the probe nucleic acid fragment. The treatment of the surface of the solid support may be performed by a method utilizing a silane coupling agent containing an amino group, aldehyde group, epoxy group or the like. In the surface treatment using such a silane coupling agent, the amino group, aldehyde group or the like are fixed to the solid support surface via a covalent bond, so that the cDNA or PCR product can be fixed to the support surface more stably than in the case of surface treatment with a poly-cationic compound.
As a variation of the above method utilizing the charge of the probe nucleic acid fragment, a method has been reported where a PCR product modified with an amino group is suspended in SSC (buffer solution of standard sodium chloride/citrate), and the suspension is spotted onto the surface of sililated slide glass and, after incubation, a processing with sodium borohydride and a heat processing are successively performed. In this method, however, the probe nucleic acid fragment cannot be fixed with sufficient stability.
(2) In the case where the fixed probe nucleic acid fragment is a synthetic oligonucleotide, initially an oligonucleotide to which a reactive group has been introduced is synthesized. The oligonucleotide is then spotted onto a solid support whose surface has been treated such that a reactive group is formed, so that the oligonucleotide is bound and fixed to the solid support surface via covalent bonding. For example, in one method, an amino-group introduced oligonucleotide is reacted with a slide glass onto a surface of which an amino group has been introduced, under the presence of PDC (p-phenylene diisothiocyanate). In another method, an aldehyde-group introduced oligonucleotide is reacted with the slide glass. These two methods are advantageous over the fixing method (1) based on static bonding where electric charge of the DNA fragment is utilized, in that the oligonucleotide is fixed to the surface of the solid support in a stable manner. However, these methods have problems. For example, in the method involving the presence of PDC, the reaction between PDC and the amino-group introduced oligonucleotide is slow. In the method utilizing the aldehyde-group introduced oligonucleotide, the stability of the Schiff base which is a reaction product, is low (and therefore hydrolysis is likely to occur).
A technique has been proposed recently in which an oligonucleotide analog called PNA (peptide nucleic acid) is used instead of an oligonucleotide or polynucleotide (including synthetic oligonucleotide, DNA fragment and RNA fragment) as the probe nucleic acid fragment in a DNA chip. A method of fixing the PNA to the solid support via covalent bonding is known from Japanese Patent Application Laid-Open (kokai) No. 11-332595 where the combination of avidin and biotin is used. This publication discloses the use of a surface plasmon resonance (SPR) sensor as the solid support.